The developing fetal heart grows primarily by proliferation of all cell types, including cardiomyocytes. In sheep, cardiomyocytes gradually cease dividing and become binucleated (terminal differentiation) beginning at 100 days gestation of a 145 day gestational period. Once cardiac myocytes terminally differentiate they can no longer divide but they retain an enormous capacity to enlarge. This maturation step occurs mostly before birth in sheep. The regulation of cardiomyocyte replication, before the cells lose their generative capacity, is important, but the mechanisms that determine: 1) how many cells are optimal and 2) when a cell should stop dividing, are not known. We discovered that thyroid hormone, in the form of 3,3',5-tri-iodo-L-thyronine (T3), is a powerful inhibitor of proliferation in cardiomyocytes from135 day old sheep fetuses in vitro. In late gestation, cortisol stimulates the conversion of the less potent thyroxine (T4) to the more potent T3; thereafter, T3 levels begin to increase. Thus, because of this coincidental timing, T3 has become a candidate for the most powerful regulator of the near term maturation of the myocardium. It may also be a culprit in terminating proliferation long before the heart has generated its optimal number of cardiomyocytes. Thus, T3 regulation has clinical relevance for the relatively common conditions when maternal thyroxine levels are outside the normal range. In order to determine the degree to which T3 regulates the cell cycle in working myocytes, we request exploratory funds to carry out two aims over two years. Aim 1 will determine the degree to which thyroid hormone regulates cardiomyocyte proliferation and maturation of cardiomyocytes in normally growing late gestation sheep fetuses (125-130 days; term ~145 days). T3 will be administered to both intact and thyroidectomized fetal sheep to determine the changes in cardiomyocyte replication caused by low, normal, and high T3 circulating concentrations at an age ~10 days earlier than the normal T3 surge. Cardiomyocyte proliferation will be monitored using Ki-67 and BrdU as markers. Expression levels of deiodinases and thyroid receptors will also be measured; levels of key cell cycle proteins will be analyzed by western blot. Aim 2 will determine the roles of alternative signaling pathways that are stimulated by T3 in vitro and lead to inhibition of cell cycle activity. The importance of the MAPK and PI3K signaling cascades in regulating proliferation under the influence of T3 will be evaluated by measuring the activation levels of ERK, p38, JNK, AKT, mTOR and p70S6K, as well as key cell cycle proteins. So-called non-genomic pathways will also be evaluated. Expression levels of deiodinases and thyroid receptors will be measured. This study offers the opportunity to determine whether thyroid hormone is a powerful suppressant of proliferation in the immature cardiac myocyte and on the maturation of the myocardium. Once completed this should also indicate the degrees to which classical and non-classical signaling pathways regulate the T3 stimulated changes in cardiomyocyte behavior. A positive outcome will lead to further work in animal and human populations. PUBLIC HEALTH RELEVANCE: This study will determine the relevance of fetal T4 and T3 levels in regulating the proliferation of working cardiomyocytes before birth. Because maternal thyroid hormones cross the placenta and influence fetal levels, maternal thyroid disease may seriously affect heart cardiomyocyte endowment. Low cardiomyocyte numbers could lead to a myocardium that is disadvantaged for the work it will perform in extrauterine life. . [unreadable] [unreadable] [unreadable]